CN113564230A - In-situ detection method for circular RNA - Google Patents
In-situ detection method for circular RNA Download PDFInfo
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- CN113564230A CN113564230A CN202110853547.6A CN202110853547A CN113564230A CN 113564230 A CN113564230 A CN 113564230A CN 202110853547 A CN202110853547 A CN 202110853547A CN 113564230 A CN113564230 A CN 113564230A
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6841—In situ hybridisation
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C12Q1/682—Signal amplification
Abstract
The invention discloses an in-situ detection method of circular RNA, which realizes the amplification of signals through rolling circle amplification, and is characterized in that a target nucleic acid molecule to be detected is converted into a circular nucleic acid molecule which is specifically corresponding to the target nucleic acid molecule to be detected. The method specifically hybridizes two sections of regions of a padlock probe formed by a single-stranded DNA with a target sequence of target nucleic acid, when bases at two ends of the probe are completely complementary with the target sequence, DNA ligase connects two ends of the probe to form a circular DNA molecule, the circular DNA molecule is amplified through rolling circle amplification, and finally, the detection probe is used for detecting an amplification product to realize the detection of the target RNA. The invention can detect the existence and distribution condition of the circular RNA in different tissues or different parts of the same tissue with high sensitivity.
Description
Technical Field
The invention belongs to the technical field of nucleic acid detection, and particularly relates to an in-situ detection method for circular RNA.
Background
Circular RNA (circular RNA) is a type of circular RNA molecule that is formed by alternative splicing during transcription, and that is joined end-to-end. Compared to its linear transcript, circular RNA is more stable and less prone to degradation by RNases because it has no 5 'and 3' ends. There is increasing evidence that circular RNA plays an important role in a number of important diseases and cancers.
Nucleic acid in situ detection techniques enable localized detection of DNA and RNA in cell and tissue samples. The traditional in situ detection technology of nucleic acid is the in situ hybridization technology (ISH), the method is to hybridize the labeled detection probe and the target nucleic acid, the unbound probe is washed away, the detection probe specifically hybridized with the target nucleic acid is detected by the label, thereby realizing the detection of the target nucleic acid. Wherein the single-molecule fluorescence in situ hybridization technique (smFISH) enables in situ detection of a single RNA molecule by hybridizing multiple detection probes on the single RNA molecule to obtain a sufficiently strong signal. Compared with the traditional ISH, the single-molecule fluorescence in situ hybridization technology has higher sensitivity and specificity. However, due to the limitations of the principle of the technology itself, the smFISH technique cannot distinguish linear RNA transcripts from circular RNA of the same gene, and is therefore not suitable for in situ detection of circular RNA.
Currently, a circular RNA detection kit commonly used in domestic laboratories is an in situ hybridization kit of BaseScope of ACD corporation in the united states, which is developed and applied based on the principle of Branched DNA technology (bDNA). The bDNA technology is characterized in that a pair of double Z-shaped probes is hybridized to a target sequence, signals are amplified step by step, and finally a red chromogenic substrate is adopted for detection. Single RNA transcripts appear as red dot-like or cluster-like signals.
Disclosure of Invention
The invention aims to provide an in-situ detection method for circular RNA.
The technical scheme of the invention is as follows:
an in-situ detection method of circular RNA comprises the following steps:
(1) designing at least one lock-type probe, wherein the at least one lock-type probe is specifically hybridized and complemented with the circular RNA to be detected in the sample to be detected;
(2) filling nucleotides along the circular RNA by a locking probe which is specifically and complementarily hybridized with the circular RNA through DNA ligase which can take the RNA as a template to connect DNA, and obtaining at least one circular template;
(3) performing rolling circle amplification by using the at least one annular template as an amplification template to obtain at least one rolling circle amplification product;
(4) hybridizing the at least one rolling circle amplification product with at least one detection probe to carry out in-situ detection on the circular RNA.
In a preferred embodiment of the present invention, the sample to be tested is a cultured cell, a cell in a tissue or a cell in a tissue section.
Further preferably, the sample to be tested is a cell in a tissue section.
In a preferred embodiment of the invention, the DNA ligase is splntr ligase.
In a preferred embodiment of the invention, the enzyme used for rolling circle amplification is phi29 polymerase.
In a preferred embodiment of the invention, the detection probe is modified with a fluorescent label, a chromogenic label, an enzymatic label, a radioactive label, a magnetic label or a luminescent density label.
In a preferred embodiment of the invention, the at least one lock probe comprises natural and/or non-natural nucleotides.
In a preferred embodiment of the present invention, the sample to be tested is a cell in a tissue section, the DNA ligase is SplintR ligase, and the enzyme for rolling circle amplification is phi29 polymerase.
Further preferably, the detection probe is modified with a fluorescent label, a chromogenic label, an enzyme label, a radioactive label, a magnetic label or a luminescent density label.
Still further preferably, the at least one lock probe comprises natural and/or non-natural nucleotides.
The invention has the beneficial effects that:
1. the invention realizes the amplification of signals through rolling circle amplification, and the premise is that a target nucleic acid molecule to be detected is converted into a circular nucleic acid molecule which is specifically corresponding to the target nucleic acid molecule to be detected. The method specifically hybridizes two sections of regions of a padlock probe formed by a single-stranded DNA with a target sequence of target nucleic acid, when bases at two ends of the probe are completely complementary with the target sequence, two ends of the probe are connected by DNA ligase to form a circular DNA molecule, then the circular DNA molecule is amplified through Rolling Circle Amplification (RCA), and finally the detection of the target RNA is realized by detecting an amplification product by using a detection probe.
2. The invention can detect the existence and distribution condition of the circular RNA in different tissues or different parts of the same tissue with high sensitivity. Compared with the existing BaseCope technology, the detection steps are reduced, special instruments and equipment are not needed, the detection time is shortened, the operation is simple, and the performance is stable.
Drawings
Fig. 1 is a schematic view of the principle of embodiment 1 of the present invention.
FIG. 2 is a graph showing the results of detection in example 1 of the present invention.
FIG. 3 is a graph comparing the effects of detecting Cdr1as in example 2 and the BaseCope according to the present invention.
Detailed Description
The technical solution of the present invention will be further illustrated and described below with reference to the accompanying drawings by means of specific embodiments. Example 1 mouse cerebellum for experimental sample review the padlock probe method for in situ detection of circular RNA Cdr1as and Phf21 a.
The principle of this embodiment is shown in fig. 1, and specifically includes the following steps:
preparing a tissue sample:
the brain of C57 wild-type mice (Xiamen university medical school) was dissected and fixed in 4% Paraformaldehyde (PFA) overnight at 4 ℃ and then transferred to 30% sucrose in 1 XDEPC-PBS for dehydration overnight. They were embedded in an Optimal Cutting Temperature (OCT) compound at-80 ℃ and frozen, and frozen sections of 15 μm were prepared using a Leica cryostat (Cat No. CM1905; Leica, Germany). All tissue section slides were stored at-80 ℃ prior to use.
(II) tissue sample pretreatment:
mouse brain sections on slides were pre-fixed in 4% (w/v) PFA-DEPC-PBS for 5min at Room Temperature (RT) and then in DEPC-PBS-T (0.05% Tween-20(Sigma) in DEPC-treated PBS). Then permeabilized with 0.1MHCl (Sigma) for 5min at room temperature. After washing twice in DEPC-PBS-T, the slides were immersed in 70% (v/v), 85% (v/v) and 100% ethanol (VWR) for 2min each and air dried. Finally, a Secure-Seal hybridization chamber (Thermo Scientific) of 9 mm diameter and 0.8 mm depth was attached to the surface on which the sample was placed.
(III) padlock probe hybridization and rolling circle amplification:
the sequences of the padlock probe, rolling circle amplification primer and detection probe used in this step are shown in Table 1.
(1) Hybridization of probes
To a 1.5mL centrifuge tube in sequence was added hybridization buffer at a final concentration of 6XSSC, 10% formamide (Sigma), at a final concentration of 0.2. mu.M padlock probe, supplemented with DEPC-H2And O is added to 50 mu L, evenly mixed and centrifuged, and then added into the reaction hole, and the mixture is placed into a constant temperature incubator at 37 ℃ for reaction for 2 hours. After the reaction was completed, the reaction mixture was washed 3 times with DEPC-PBS-T.
(2) Probe looping
50 μ L of the ligation mix contained 1 XSplintR ligase reaction buffer (NEB), 0.5U/ml SplintR ligase (NEB), 1U/ml RiboLock RNase Inhibitor (Thermo Scientific), 0.2mg/ml BSA (NEB), supplemented with DEPC-H2O to 50 μ L, added to the well chamber and incubated at 37 ℃ for 1 h. After the reaction was complete, the unligated padlock probe and enzyme were removed by washing 3 times with DEPC-PBS-T. Add hybridization buffer with final concentration of 6XSSC, 10% formamide, 0.2. mu.M rolling circle amplification primer to 50. mu.L reaction system, supplement DEPC-H2O to 50. mu.L, reacted at 37 ℃ for 30min, and then washed 3 times with DEPC-PBS-T.
(3) Rolling circle amplification
50 μ L of RCA mixture containing 1 XPhi 29 DNA polymerase buffer (Thermo Scientific), 5% glycerol, 1mM dNTP (Thermo Scientific), 1U/ml phi29 polymerase (Thermo Scientific), 1U/ml RiboLock RNase Inhibitor, 0.2mg/ml BSA was added to the reaction wells and incubated overnight at room temperature. Then washed 3 times with DEPC-PBS-T.
(4) Detection probe hybridization and image acquisition
And hybridizing the rolling circle amplification product with a detection probe, and finally detecting after cell nucleus staining by using DAPI. The method comprises the following specific steps: 50 μ L of a mixture containing 20% formamide in 2 XSSC, 100nM fluorescently labeled detection probe was prepared and incubated at 37 ℃ for 30 min. After the reaction was completed, the reaction site was marked by a circle and removed after washing 3 times with DEPC-PBS-TThe Secure-Seal hybridization chamber dehydrates slides in 70%, 85% and 100% gradient ethanol for 2min each, and air dries. Finally, 100ng/mL DAPI is added
Gold antipade mount block (Fermentas) was incubated at room temperature for 10min before fluorescent microscopy and photographed. As shown in FIG. 2, Cdr1as and Phf21a are highly expressed in the granular layer of cerebellum. The upper half is a global map of the whole cerebellum with a scale of 500 μm, the lower half is a local map with a scale of 200 μm. .
Example 2 mouse cerebellum for experimental samples circular RNA Cdr1as was detected in situ comparing the padlock probe method and BaseScope method.
In this example, the procedure for lock-probe detection of Cdr1as was the same as in example 1, and the commercial BaseScope kit was completed according to the manufacturer's instructions. The comparative detection result of this example is shown in fig. 3, and it can be seen that the detection efficiency of both methods is very high in the detection of the Cdrlas gene.
Table 1 all probe sequences used in examples 1 and 2:
marking: a sequence complementary to the loop-forming junction of the circular RNA to be detected; thickening: detecting the hybridization site of the probe and the rolling circle amplification primer.
All purchased padlock probes were not phosphorylated. Prior to use, phosphorylation was performed as follows: mu.M padlock probe, 1 XPNK buffer A (Thermo Scientific), 1mM ATP (Thermo Scientific), 0.2U/. mu. L T4 polynucleotide kinase (Thermo Scientific) were mixed well and incubated at 37 ℃ for 30min, then the enzyme was inactivated at 65 ℃ for 10 min. Phosphorylated padlock probes can be stored at-20 ℃ until use.
The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims.
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Claims (10)
1. A method for in-situ detection of circular RNA, which is characterized in that: the method comprises the following steps:
(1) designing at least one lock-type probe, wherein the at least one lock-type probe is specifically hybridized and complemented with the circular RNA to be detected in the sample to be detected;
(2) filling nucleotides along the circular RNA by a locking probe which is specifically and complementarily hybridized with the circular RNA through DNA ligase which can take the RNA as a template to connect DNA, and obtaining at least one circular template;
(3) performing rolling circle amplification by using the at least one annular template as an amplification template to obtain at least one rolling circle amplification product;
(4) hybridizing the at least one rolling circle amplification product with at least one detection probe to carry out in-situ detection on the circular RNA.
2. The method for in situ detection of circular RNA according to claim 1, wherein: the sample to be detected is cultured cells, cells in a tissue or cells in a tissue section.
3. The method for in situ detection of circular RNA according to claim 2, wherein: the sample to be detected is a cell in a tissue section.
4. The method for in situ detection of circular RNA according to claim 1, wherein: the DNA ligase is SplintR ligase.
5. The method for in situ detection of circular RNA according to claim 1, wherein: the enzyme used for the rolling circle amplification is phi29 polymerase.
6. The method for in situ detection of circular RNA according to claim 1, wherein: the detection probe is modified with a fluorescent marker, a chromogenic marker, an enzyme marker, a radioactive marker, a magnetic marker or a luminous density marker.
7. The method for in situ detection of circular RNA according to claim 1, wherein: the at least one lock probe contains natural and/or unnatural nucleotides.
8. The method for in situ detection of circular RNA according to claim 1, wherein: the sample to be detected is cells in a tissue section, the DNA ligase is SplintR ligase, and the enzyme used for rolling circle amplification is phi29 polymerase.
9. The method for in situ detection of circular RNA according to claim 8, wherein: the detection probe is modified with a fluorescent marker, a chromogenic marker, an enzyme marker, a radioactive marker, a magnetic marker or a luminous density marker.
10. The method for in situ detection of circular RNA according to claim 9, wherein: the at least one lock probe contains natural and/or unnatural nucleotides.
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| CN114854736A (en) * | 2022-06-23 | 2022-08-05 | 香港中文大学（深圳） | Circular nucleic acid molecule, method for producing same, nucleic acid probe and detection method |
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Cited By (2)
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| CN114854736B (en) * | 2022-06-23 | 2023-12-15 | 香港中文大学（深圳） | Circular nucleic acid molecule, method for producing same, nucleic acid probe, and method for detecting same |
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